The major barrier in achieving an HIV-1 cure is the rapid establishment of latent infection. One strategy to target latently infected cells in a cure strategy is to induce a deep latency, avoiding virus rebound when anti-retroviral therapy (ART) is removed. Several lines of evidence now show in latently-infected cells that the mTORC1 complex, a master regulator of autophagy, a major metabolic process that maintains host cell physiology, severely restricts the reactivation of HIV-1. In this work we hypothesized that the induction of autophagy will induce a deep latency and suppress the reactivation of HIV-1 from latently-infected CD4+ T cells. Our experimental procedures consist of treating latently infected cells (GFP expressing J-lat 10.6 cells) with FDA-approved autophagy-inducing drugs (i.e., anti-depressants such as paroxetine, promethazine, trimipramine) and then reactivating cells using a selection of latency-reversing agents (LRAs), followed by the evaluation of levels of the provirus using GFP expression as a readout by flow cytometry. RESULTS: The autophagy-inducing drug, paroxetine, attenuated virus reactivation 2.3-fold using PMA and prostratin as the LRAs (p=0.0091, t-test), but not with TNF-α and SAHA, that lead to reactivation of proviral DNA through other mechanisms. These data suggest that paroxetine intersects with the PKC pathway to modulate virus reactivation. Although we show that paroxetine induces autophagy in these cells, further work will allow us to determine if the block to latency reversal we observe is achieved via this mechanism. Considering that the dysregulation of autophagy influences both the pathological presentation of HIV-1-associated neurodegenerative disease and reactivation from latency, this work has the potential to inform on therapeutics for HAND and functional cure strategies by repurposing FDA-approved drugs for HIV-1.

Background: Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of infectious and non-infectious pulmonary diseases, suggesting that their lung immunity is not fully restored. Cytotoxic CD8 T-cells are essential in controlling chronic viral infections. However, inappropriate and excessive CD8 T-cell activation during HIV infection can have serious adverse effects associated with lung mucosal tissue damage. Furthermore, while tobacco smoking is part of the lifestyle of many Canadians, this changes the lung environment and promotes pulmonary inflammation. We thus aimed to characterize the effects of HIV and smoking on tissue-resident memory (Trm) CD8 T-cell dynamics in the human lung.

Methods: Bronchoalveolar lavage (BAL) fluid and matched blood were obtained from asymptomatic ART-treated (median undetectable viral load: 8 years) HIV+ smokers and non-smokers and uninfected smokers and non-smokers (n=6 per study group). Lymphocytes were isolated for flow cytometric analysis.

Results: Both smoking and positive HIV status were independently associated with significantly higher total CD8 T-cell frequencies in BAL. Two distinct CD8 Trm subsets were observed based on CD103 expression (CD103+CD69+CD49a+, CD103-CD69+CD49a+). Furthermore, smoking, but not HIV status, was associated with higher CD103+CD8 Trm within different study groups. Chronic HIV infection was also associated with higher frequencies of CD8 non-Trm (CD103-CD69-CD49a) co-expressing KLRG1/CX3CR1, suggesting increased CD8 T-cell infiltration from the circulation into the lung mucosa. Moreover, CD8 T-cells from HIV+ versus HIV- study participants displayed higher levels of cytotoxic effector molecules granzymes A and B.

Conclusions: Despite long-term ART, chronic pulmonary inflammation caused by HIV infection may dysregulate mucosal CD8 T-cell cytolytic functions and cause infiltration of pro-inflammatory CD8 T-cells into the lung mucosa. Smoking could promote CD8 T-cell retention in the lung via upregulation of CD103 on these cells.

Background: People living with HIV (PLWH) develop cardiovascular disease (CVD) at higher rates than age-matched uninfected persons. Aside from classic risk factors for CVD, co-infection with cytomegalovirus (CMV) may have a substantial effect on the progression of atherosclerosis and cardiovascular risk. CMV infection drives the expansion of NKG2C+CD57+ adaptive NK (adapNK) cells with memory-like properties. We questioned whether the frequency of these cells was associated subclinical atherosclerosis.

Methods: 194 participants aged ≥40 yrs enrolled in the CHACS were included, (n=128 CMV+PLWH, 8 CMV-PLWH, 37 CMV mono-infected and 21 double negatives). All underwent coronary angiography by computed tomography, and total coronary plaque volume (TPV) was obtained. All were free of overt CVD. All PLWH were on-ART for a mean duration of 15 yrs. Subclinical atherosclerosis was defined as absent if TPV=0, and as present if TPV>0. We categorized NKG2C+CD57+ adapNK cells frequency as low if <4.5%, intermediate if between 4.6% and 20% and high if >20%.

Results: The frequency of NKG2C+CD57+ adapNK cells was higher in CMV+ (16.3 [6.6-36.7]) than CMV- participants (2.5 [0.9-3.0]), (p<0.0001, Mann Whitney tests). A greater proportion of CMV+PLWH with high adapNK cell frequencies had absence versus presence of subclinical atherosclerosis [61.90% versus 39.53%, p=0.03, Chi-square test) with a similar trend in CMV mono-infected participants [46.15% versus 34.78%]. In an independent Poisson regression analysis, after adjusting for classic cardiovascular risk factors, CMV+ persons with high adapNK cell frequencies had a relative risk (RR) for the absence of coronary artery atherosclerosis of 0.75 (95% CI, 0.58-0.97, p= 0.03). Increasing age and smoking intensity increased the RR for atherosclerosis. HIV infection had no effect on the RR of atherosclerosis (RR, 1.08, 0.81-1.42, p= 0.58).

Conclusion: High frequencies of NKG2C+CD57+ adapNK cells are associated with a reduced risk of atherosclerosis in CMV+PLWH and CMV mono-infected individual.

Background: The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV vaccine and targeting the conserved regions of HIV-1 envelope (Env) are very promising at optimizing the HIV-specific immune responses.
Objective: Infusion of the highly conserved elements (CE) of HIV envelope glycoprotein including the 9 highly conserved elements (9CE) in HIV gp120 or the Membrane-Proximal External Region (MPER) on the gp41, with the DC-targeting domains of EboGP (EboGPΔM), and test their ability to target DCs/Macrophages and to induce immunes responses in mouse.
Methods: To produce VLPs, we transfected 293T cells with EboGP∆M-9CE, EboGP∆M-MPER or HIV Env plasmids, packaging plasmid (Δ8.2) and together with or without a multiple-gene deleted HIV-based vector encoding a Gaussia luciferase gene (ΔRI/ΔEnv/Gluc). Female BALB/c mice were immunized subcutaneously with 100 ng P24 of EboGP∆M-MPER, EboGPΔM-9CE, HIV Env-VLPs or PBS on days 0, 21 and 56. At days 21, 34, and 76 of post-immunization, the mice blood was collected and the anti-HIVgp41, -HIVgp140, -HIVp24 and -EboGP specific antibodies were measured by corresponding ELISA.
Results: Data showed that the EboGPΔM-9CE or EboGPΔM-MPER was expressed in the cells and incorporated into VLPs that can efficiently target a human monocyte cell line (THP-1) and human monocyte-derived macrophages (MDMs). Animal studies revealed that immunization with VLPs containing the above chimeric proteins, especially EboGPΔM-MPER, induced significantly higher anti-HIV Env antibodies than HIV-Env-VLPs alone in mouse serum. Furthermore, this study suggested that EboGP∆M-MPER has induced not only a more effective immune response to MPER but also enhance the immune response against other HIV components, such as HIV Gag.
Conclusion: EboGP∆M-MPER pseudotyped HIV VLPs significantly enhanced HIV-specific immune responses and represented as a potential vaccine candidate.

Background: COVID-19 and influenza are both highly contagious respiratory diseases with a wide range of severe symptoms and cause great disease burdens globally. It has become very urgent and important to develop a bivalent vaccine that is able to target these two infectious diseases simultaneously.
Objective: We generated several rVSV bivalent vaccine candidates that co-expressed SARS-CoV2 Delta variant spike protein (SP) or RBD and four copies of highly conserved influenza M2 ectodomain (M2e) fused with a DC-targeting/activation domain derived from EBOV GP (EboGPΔM) based on our previously reported vaccine platform. Here, we characterized the expression of SARS-CoV-2 Delta variant spike protein (SP) or RBD and influenza M2 ectodomains of these bivalent vaccine candidates and their abilities to induce immune responses against SARS-CoV-2 SP, especially Delta SP, and influenza M2e.
Results: Our studies showed that in contract to the VSVwt, these VSV biovalent vaccines showed much attenuated replication kinetics had no or much milder cytopathic effects in most tested cell lines. Animal studies have shown that immunization with these bivalent rVSV vaccines induced efficient humoral and cell-mediated immune responses against both SARS-CoV-2 and influenza M2e protein. Significantly, our vaccine candidates induced production of high levels of neutralizing antibodies that protected cells against SARS-CoV-2 Delta and other SP-pseudovirus infections in culture. Furthermore, vaccination with the bivalent VSV vaccine via either intramuscular or intranasal route efficiently protected mice from the lethal challenge of H1N1 and H3N2 influenza viruses and significantly reduced viral load in the lungs.
Conclusion: These studies provide convincing evidence for the high efficacy of this bivalent vaccine to prevent influenza replication and initiate robust immune responses against SARS-CoV-2 Delta variants. Further investigation of its efficacy to protect against SARS-CoV-2 Delta variants will open a new avenue to control two contagious respiratory infections, COVID-19 and influenza

Background: Accurate information on HIV diversity, subtype distribution and drug resistance is critical to guide HIV treatment and curative strategies in resource limited settings. In West African,Ghana, highly diverse HIV strains, including complex recombinants, co-circulate, but the full extent of this diversity is incompletely understood. A well-organized government sponsored HIV/AIDS treatment in Ghana began in the year 2003 with the most recent WHO-compliant Ghanaian HIV drug resistance survey occurring between 2004-2013. Towards addressing these gaps, we are characterizing plasma HIV Protease/Reverse Transcriptase and Integrase sequences in Ghanaian participants, as well as full-genome HIV sequences where possible, to better understand the transmitted/acquired drug resistance mutation burden and molecular epidemiology of HIV in the region.

Methods: We used nested RT-PCR and a combination of Sanger and Illumina sequencing to characterize HIV sequences from a preliminary set of 55 plasma samples from Ghanaian participants aged 16 and over. Forty-eight persons (87%) were treatment naive at time of sample collection, while 7 persons (13%) had discontinued therapy.

Results: To date, Protease/Reverse Transcriptase and Integrase sequencing was successful for 47 (85%) and 35 (64%) samples, respectively. Of these, 14 participants, including 9 treatment-naïve, 3 treatment-experienced participants and 2 persons whose treatment history was unknown, respectively, harbored at least one drug resistance mutation. The major NNRTI-resistance mutations RT-K103N (n=2), RT-K101E (n=1) and RT-Y188L (n=1) were observed. Whole genome sequences, available for 25 participants to date, revealed 11 (44%) CRF02_AG, one (4%) complex recombinant form 06_cpx, 2 (8%) putative novel recombinants comprised of CRF02_AG and subtype A1, and 11 putative complex recombinants comprising at least 3 subtypes.

Conclusions: Our observations are consistent with extensive HIV diversity in Ghana, which facilitates the ongoing creation of new complex recombinant forms. Study findings will improve understanding of the Molecular Epidemiology of HIV and drug resistance mutation prevalence, in Ghana.

Background: Invariant Natural Killer T (iNKT) cells are innate lymphocytes critical in combatting viral infection by bridging the innate and adaptive immune systems. Our lab showed in HIV infection, expression of lymphocyte activation gene 3 (LAG-3), an inhibitory immune checkpoint marker, is increased on iNKT cells and correlated with decreased functionality. Another checkpoint molecule, program cell-death-1 (PD-1), is shown to be increased on iNKT cells in HIV infection, correlating to decreased function. LAG-3 and PD-1 expression kinetics and relationship to iNKT cellular function is not well characterized, and it is unknown whether blocking LAG-3 alone, or in conjunction with PD-1, will restore iNKT function and immune effectiveness.

Methods: Utilizing peripheral blood mononuclear cells from HIV-uninfected donors (n=4), iNKT expression of LAG-3 and PD-1 was assessed via a multi-day stimulation (24hr, 48hr, 4, 7, 10 day). Efficacy of anti-LAG-3 and anti-PD-1 antibody blockades was assessed via a 10-day assay, with enhanced proliferation as the main outcome monitored.

Results: Percent and median fluorescence intensity (MFI) of both LAG-3 and PD-1 peaked at Day 7 (LAG-3: 88.5%, 6163.8 MFI; PD-1: 80.5%, 7731.8 MFI), with a steep decrease by Day 10, when iNKT proliferation was at its peak. In the presence of the anti-LAG-3 or anti-PD-1 antibody blockades, there was a 14-fold increase and 17-fold increase of the iNKT population, respectively. Combining anti-LAG-3 and anti-PD-1 blockade systems resulted in 22-fold proliferation increase.

Significance: This study is the first to characterize LAG-3 and PD-1 expression kinetics on iNKT cells and provides proof-of-concept for LAG-3 and PD-1 as immunotherapeutic targets, by restoring cellular proliferative ability. This blockade system will be applied to HIV-positive samples to assess if HIV-mediated dysregulation of iNKT function can be reversed and thereby ameliorate immune responses to various opportunistic infections, as well as boost viral control in a functional HIV cure approach.

Though antiretroviral therapy (ART) suppresses HIV replication, it is not able to eradicate virus-infected long-lived cells harboring integrated latent HIV proviruses known as “viral reservoir (VR)”. One important mechanism contributing to the survival and persistence of these VR is their capacity to prevent cell death from host immune effectors such as CD8+ cytotoxic T lymphocytes (CTLs). Latency reversing agents (LRA) can reactivate latent virus to enhance their immune recognition but are inefficient at sensitizing reactivated cells to death. Recent evidence reveals that latent HIV infected cells have an intrinsic resistance to cell death responses modulated by the pro-survival protein BCL-2. Transcriptomic analysis of CTL-resistant CD4+ T cells revealed BCL-2 upregulation is one of the strategies exploit by the virus to prolong latent cell survival. Previously, we showed that a novel bivalent SMAC mimetic (SM)) is an effective LRA, which reactivates latently infected CD4+ T cells but induces a modest reduction of VR in humanized mice. As HIV-infected cells also upregulate BCL-2 expression, the ability of these compounds to sensitize VR to cell death remains limited. Here-in, using established HIV infection models, we demonstrate a novel approach to augment the cell death and elimination of latently HIV infected cells with BCL-2 inhibitor alone and/or in combination with SM treatment. Our results reveal that BCL-2 inhibitor alone is not sufficient to drive reactivation of latently HIV infected cells but showed a selective toxicity to latently HIV infected cells. Importantly, combination of SM treatment followed by BCL-2 inhibition show an additive effect in the reduction of latently infected cells via the activation of apoptotic cell death. Overall, these results provide the rationale to evaluate the therapeutic potential of combining LRAs with pro-survival protein antagonists in the context of VR elimination strategies in humanized mice preclinical models.

Despite being a relatively simple virus with only one viral envelope glycoprotein (Env), HIV has caused one of the largest pandemics in history. HIV Env is highly glycosylated, well shielded from most humoral responses and is in relatively low abundance on virions (~8-14 spikes), which collectively, complicates strategies targeting this molecule on the virus. While Env is sparse on the HIV surface, numerous cellular proteins acquired from the host membrane during budding can decorate the HIV surface. Notably, some cellular proteins can be in excess of Env, suggesting that they could serve as attractive targets for therapeutic strategies. Understanding the biology of these virion-incorporated cellular proteins in HIV infection is critical since some host proteins can impact virion infectivity and homing. Previous studies of host proteins on HIV have traditionally employed bulk techniques which assess the average of virus populations instead of individual virions. Our work uses an emerging technique called flow virometry (FV) to characterize the surface of virions and quantify virion-incorporated proteins on individual virus particles. Importantly, our single virion analyses are conducted without additional methods to enhance virus detection (concentration, coupling to magnetic beads, etc.), removing additional biases that these methods can add. Notably, using fluorescence reference beads and calibration software, we provide estimates of total proteins present on individual virions, which are in line with data reported from traditional methods. Finally, in an effort to adapt this technique for phenotyping viruses from clinical samples, we have generated initial success in staining proteins of interest on virions in human plasma and sera. These findings highlight the utility of FV to phenotype virions, which may be useful in clinical applications or in identifying new targets on HIV for alternative therapeutic strategies.

Introduction: Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal monocyte-derived macrophages (MDMs). Degradation of IAPs by synthetic second mitochondrial activator of caspases (SMAC) mimetics (SMs) induce cell death of HIV-Vpr-treated MDMs. Herein, we hypothesized that SMs selectively induce programmed cell death of HIV-infected MDMs.

Methods: U937 and U1 cell lines, in vitro mock and HIV-1 (clinical isolate CS204) or HIV-1 NL4.3-Bal-HSA (heat stable antigen reporter) infected MDMs from healthy donors, and MDMs derived from HIV-infected patients were treated with SMs or IAP silencing RNA. Cell death was assessed by intracellular PI staining or Annexin V staining and flow cytometry. Degradation of IAPs and proteins involved in apoptosis such as RIPK1, caspase-3, -8-, and -9 were assessed by western immunoblotting. HIV-1 DNA were quantified through nested qPCR.

Results: SMs induced apoptosis in U1, in vitro HIV-infected MDMs, and MDMs derived from HIV-infected patients, but not in uninfected U937 and mock-infected MDMs. Moreover, apoptosis specifically occurred in HIV-infected MDMs expressing HSA reporter protein, as shown by the activation of caspase-3, -8, and -9. In healthy MDMs, SM-induced IAP degradation failed to induce apoptosis but with concomitant inactivation of RIPK1 by necrostatin-1 resulted in high degree of cell death. Interestingly, in vitro HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation over time, and HSA-expressing HIV-infected MDMs showed decreased levels of RIPK1 compared to mock or uninfected MDMs.

Conclusion: Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV-1 possibly through the concomitant downregulation or inactivation of RIPK1. Thus, modulation of the IAP and apoptosis pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

Background: HIV antiretrovirals (ARVs) can damage mitochondria and affect their function. The relatively new drug class of HIV integrase strand transfer inhibitors (InSTI) are popular among people living with HIV for its high tolerability, few side-effects, and low pill burden. However, less is known about their mitochondrial toxicities compared to older ARVs. The InSTI dolutegravir has been associated with weight gain in adults, which may reflect changes in cellular metabolism governed by mitochondria. Mitochondrial toxicity of recently approved InSTIs bictegravir, elvitegravir+cobicistat, and cabotegravir remain unclear.

Our aim was to characterize the effects of InSTI exposure in cultured immune cells on mitochondrial health, cellular activation, and proliferation.

Methods: PBMCs from healthy volunteers were activated with anti-CD3/CD28 for 6 days while exposed to 1xCmax InSTIs in 0.1% DMSO. Mitochondrial intermembrane potential (MMP), reactive oxygen species (mtROS), and mass (mtmass), along with cellular proliferation, apoptosis, differentiation, and activation were determined by flow cytometry. Significance was determined using paired t-tests.

Results: Compared to DMSO (n=9 biological replicates), bictegravir exposure had the most pronounced effect, with greatly decreased mtmass (p<0.001), mtROS (p<0.001), MMP (p<0.001) and arrested proliferation (p<0.001). Elvitegravir+cobicistat also decreased MMP (p<0.001) and proliferation (p<0.001). In contrast, dolutegravir and cabotegravir both increased MMP (p<0.045), while raltegravir had no effect on any parameters. In a pilot experiment, bictegravir exposure appeared to elevate early and middle but decrease late cellular activation markers compared to controls and other InSTI treatments.

Discussion: These data clearly show that InSTIs can affect mitochondria in PBMCs. Furthermore, the effects of bictegravir ex vivo suggest a potential underlying metabolic mechanism which could hinder immune responses. It is imperative to investigate the effect of InSTIs as certain toxicities may not be apparent nor revealed by clinical trials but may exert long-term immunological and health consequences.

Introduction: Gene therapy using a combination of antiviral RNAs has strong potential to cure HIV infection by providing a permanent source of HIV-resistant cells in an infected individual. shRNAs are among the top candidates for anti-HIV gene therapy because of their demonstrated specificity; our lab has previously identified an shRNA candidate targeting a highly conserved sequence in the Gag coding region of HIV RNA. shRNAs are typically transcribed from three different RNA polymerase III promoters (H1, 7SK, and U6,) that have different transcriptional efficiencies. Here, we screened several top performing shRNA candidates from the literature for efficacy and safety using the three promoters.

Methods: Preliminary efficacy was determined by measuring viral production after cotransfecting shRNAs with an NL43 HIV molecular clone in HEK 293T cells. The top-performing candidates were then cloned under H1, 7SK, or U6 and expressed from lentiviral vectors in T lymphocytes. Cells were then infected with HIV NL43 to compare replication kinetics. A competitive cell-growth assay was used to investigate whether shRNAs impact cell growth.

Results: We selected 23 shRNAs from the literature using the following criteria: high reported in vitro activity, high target site sequence conservation, and inclusion in preclinical or clinical studies. They were then cloned into plasmids with an H1 promoter. Three shRNAs expressed from the H1 promoter were particularly effective at delaying HIV replication in T lymphocytes. All shRNAs expressed from 7SK and U6 were more effective, but cell growth was negatively impacted. However, growth rates varied between different shRNA-promoter combinations, suggesting that the negative effects are partly sequence-specific.

Conclusions: 7SK and U6 promoted shRNAs are most effective for reducing HIV replication but may induce cytotoxicity depending on the sequence. Optimizing promoter-shRNA constructs and generating an effective, non-toxic combination may contribute to future stem cell transplants to functionally cure HIV infection.

Most current antivirals target discrete stages in the virus replication cycle (entry, genome replication, proteolytic cleavage of viral proteins) by inhibiting the activity of virus-encoded proteins or their interactions with host cell machinery. However, as obligate parasites, viruses are dependent upon a common subset of host cell processes for their replication. Consequently, altering the ability of a virus to effectively use a host cell process may provide an alternative strategy to impede virus replication, generating a more robust barrier to virus resistance, and affect a broader spectrum of viruses with a single agent. Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491) which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~0.25 µM), and alterations in the production of unspliced, singly-spliced and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound which impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

Susceptibility to HIV infection following sexual exposure varies greatly, suggesting underlying risk factors- such as the genital microbiome- are critical for transmission. Circumcision reduces HIV risk by 60%, however a biological explanation for this relationship remains elusive. We recently identified 6 strict anaerobic bacterial species whose abundance under the foreskin was associated with risk of HIV seroconversion, increased local levels of pro-inflammatory cytokines and tissue density of HIV target cells. These findings suggest specific anaerobes drive local inflammation thereby increasing HIV susceptibility, however our observational studies cannot elucidate causal relationships. To solve this problem, an in vitro approach is required for empirical study of foreskin-microbiome dynamics. Here we show that our newly developed 3D organotypic foreskin model recapitulates physiological properties of skin and is suitable for co-culture with relevant bacteria. Primary foreskin fibroblasts and keratinocytes are used to generate tissues with dermal and fully stratified epidermal layers that mimic fundamental skin properties such as epithelial barrier function. Dextran tracer assays show lower permeability in organotypic tissue relative fresh (ex-vivo) and cultured (7-day) foreskin explant tissues (widely used for in vitro studies but highly limited by their rapid degradation in viability and structure). When organotypic tissue was inoculated with Staphylococcus epidermidis, bacterial growth was observed by qPCR after 72 hours and tissue remained intact, suggesting that our model can support bacterial colonization. These results indicate that our model provides a stable, physiologically relevant environment for bacterial co-culture, previously absent in the literature. Our in vitro approach forms the basis for future studies to ascertain which bacterial species drive local inflammation and identify the inflammatory pathways involved, filling a current gap that has prevented the formation of causal and mechanistic conclusions. This model will deepen our understanding of foreskin-microbial interactions- a critical step toward developing novel preventative measures for HIV.

Despite the introduction of highly active antiretroviral therapy, HIV infection continues to be a major global public health issue as a chronic disease. The liver has been shown to be an HIV-infected organ causing liver disease and co-morbidity in HIV-infected individuals. We have established a model of Rhesus Macaque infected with SIV, taking the opportunity to analyze more in deep the nature of infected cells in the liver. Herein, we specifically assessed the role of CD4+ T cells.

Rhesus Macaques (RMs) were infected with the SIVmac251 (20 AID50). RMs were sacrificed at different times post-infection and cells from the liver were recovered. CD4+ T cells were stained with specific antibodies and analyzed by flow cytometry. Cell sorting was used to isolate CD4 T cells and to quantify the frequency of viral DNA and RNA.

First, we observed that the frequency of CD4 T cells was lower in SIV-infected RMs (12%) in comparison to healthy RMs (31%). After cell staining, we found that more than 40% of CD4 T cells expressed CCR5, a chemokine receptor used by SIV to infect the cells, in comparison to 8% in the blood. The majority of these cells are memory CD4 T cells (CD45RAneg and CD62Lneg). The phenotype of these cells indicated a specific phenotype of CD4 liver cells and not only a contamination from blood CD4 T cells. After cell sorting, we found that liver CD4+ T cells are infected cells. In comparison to CD4 T cells from the blood, the levels of viral DNA in the liver are similar.

Altogether, our results indicated that CD4+ T cells from the liver of SIV-infected RMs could be a possible viral reservoir. Further analyses are in progress to assess the extent of viral infection of CD4 T cells under ART.

Funding : CIHR, CANCURE, CRC program

Background: Dolutegravir (DTG) has been associated with a small increased risk of neural tube defects (NTDs) as well as weight gain and hyperglycemia. We evaluated the impact of DTG on glucose homeostasis using a mouse model.

Methods: Healthy euglycemic female C57BL/6 mice were randomly assigned to daily treatment with either control (water, N=15), 1xDTG (2.5mg/kg DTG+33.3/50mg/kg emtricitabine (E)/tenofovir disoproxil fumarate (T), N=13), yielding therapeutic levels of DTG, or 5xDTG (12.5mg/kg+33.3/50mg/kg E/T, N=15) for 8 weeks. Overnight fasted glucose, body weight, and oral glucose tolerance test (OGTT) were measured at 2, 4, 6 and 8 weeks. Fasting hyperglycemia was defined as fasting glucose >10mmol/L. Secondary outcomes included severe fasting hyperglycemia of >13.3mmol/L and OGTT glucose concentrations area under the curve (AUC). Mice were sacrificed at 9 weeks, and tissues were collected for gene expression of factors in glucose homeostasis pathways.

Results: No differences were observed in weight gain between groups. By week 6, 1xDTG animals displayed a significant increase in overnight fasted glucose. 16 of 28 animals treated with DTG (8 in 1x-DTG, 8 in 5x-DTG) had fasting hyperglycemia at least once, compared to only 1 in the control. Of the 16 DTG-treated mice, 3 had severe fasting hyperglycemia, 11 had fasting hyperglycemia at 6 weeks, and 10 returned to being euglycemic by week 8. Mice developing fasting hyperglycemia also showed higher OGTT AUC compared to controls. DTG-treated animals that remained euglycemic had a modest downregulation of hepatic genes associated with gluconeogenesis compared to controls, including glucose-6-phosphatase, phosphoenolpyruvate-carboxykinase, and PPAR-α.

Conclusions: DTG was associated with transient glucose dysregulation in some, but not all, animals. If further research shows DTG is associated with transient hyperglycemia in humans, this may partially explain the increase in NTDs seen after the rollout of DTG in Botswana, as hyperglycemia is a known risk factor for NTDs.

Integrated HIV-1 provirus in latently-infected cells represent the main barrier to an effective cure. The time of HIV-1 DNA integration (provirus ‘age’) may influence susceptibility to immune-mediated or therapeutic eradication strategies. Provirus ages can be estimated by root-to-tip regression (RTT). However, this approach relies on weak assumptions about uncertainty and variation in sequence divergence.

We obtained published HIV-1 (n=518 pre-treatment RNA and n=879 post-treatment DNA) sequences spanning vpu-env-nef from 13 seroconverters in the Zambia-Emory HIV Research Project. After screening for hypermutation, we reconstructed phylogenies with RAxML and then regressed root-to-tip distances to sampling dates for RNA sequences to root the trees. By this method, DNA sequences are dated by mapping root-to-tip distances to the regression line. In other words, it assumes that every HIV-1 sequence integrating at date T carries exactly Y mutations from the root. Our approach samples the location of the root, x-intercept (date of infection) and slope (rate of evolution) from a posterior distribution defined by a Poisson model of mutation accumulation. This Bayesian approach (bayroot) incorporates uncertainty in estimating these parameters from limited data, and allows proviral sequences to be mapped to a range of dates.

We applied both methods to date 437 HIV-1 DNA sequences. We observed significantly more variation among date estimates from RTT than bayroot, which we attribute to the fact that RTT does not account for uncertainty in locating the root or variation in the number of mutations. While including these factors in the Bayesian analysis increases model flexibility, however, they reduce variation in age estimates among DNA sequences, implying that there is insufficient information to differentiate among integration dates.

Background: Gene therapy using lentiviruses expressing RNA molecules represents a long-term treatment towards a cure for HIV infection to replace combination antiretroviral therapy and its daily administration. To effectively control HIV infection by gene therapy and avoid viral rebound, a combination of at least three antiviral RNA molecules will most likely be necessary. Classes of anti-HIV RNAs include: (1) short hairpin (sh)RNAs, which bind and cleave their viral RNA target site through the RNA-induced silencing complex; (2) aptamers which bind and inactivate a target protein or RNA through their three-dimensional structure; and (3) U1 interference (U1i) RNAs which cause either excessive splicing or inhibition of polyadenylation of a target RNA by the U1 small nuclear RNP (snRNP).

Methods: We have expressed different anti-HIV RNAs on lentiviral vectors and evaluated their activity by radioactive reverse transcriptase assays as well as cytotoxicity by competitive growth assays in a SupT1 cell line.

Results: We have shown that anti-HIV shRNAs are more potent when expressed by the RNA polymerase (Pol) III promoters U6 and 7SK in comparison to the H1 promoter due to a higher expression level. Although we have demonstrated that cytotoxicity exists when certain shRNAs are expressed from the U6 and 7SK promoter, we have discovered that replacing the loop of shRNAs with an aptamer effectively eliminates this toxicity. We also provide evidence that the combination of both anti-HIV U1i RNAs and shRNAs delays viral replication with little to no toxic effects.

Conclusion: Increased efficacy combined with decreased cytotoxicity will be most important in the development of anti-HIV and other gene therapies. Our work in optimizing the expression of anti-HIV RNAs is critical to designing the most effective combination gene therapies and has led to the discovery of a method to prevent shRNA-mediated cytotoxicity.

Currently, 38 million people are living with HIV-1. The implementation of cART inhibits HIV-1 replication but fails to eliminate latently infected cells. In macrophages, HIV-1 buds/accumulates in invaginations of the plasma membrane, termed virus-containing compartments (VCC), which are important for HIV-1 immune evasion and cell-to-cell transmission. How HIV-1 is directed to its budding site is not fully understood, but previous research in HeLa cells suggests that late endosomes / lysosomes (LELs) play a role in HIV-1 Gag trafficking. By repositioning LELs towards the juxtanuclear area, a population of Gag co-traffics with LELs, resulting in a decrease in viral release, while repositioning towards the cell periphery increases it. Downregulation of LELs proteins reduces HIV-1 release, altogether supporting LELs directing Gag towards budding sites. Here, we hypothesize that Gag hijacks LELs to direct HIV-1 assembly to VCC in infected macrophages. We used the THP-1 Gag-zip cell line, which has integrated a doxycycline-inducible HIV-1 Gag-GFP genome. By immunofluorescence, we observed at 72 hrs post dox-induction Gag-GFP accumulation in distinctive areas, which colocalized with the VCC marker CD81. Ultrastructural confirmation of VCC by electron microscopy is under way. We further evaluated LELs involvement and Rab7 or Lamp1 colocalized with Gag-GFP at VCC, but colocalization was stronger at surrounding areas. Finally, by tracking single LELs, we observed a population that co-trafficked with Gag-GFP, associated with a significant decrease in LELs tracks straightness and increase in speed variation. Here, we provide evidence that THP-1 Gag-zip macrophages resemble VCC as observed in HIV-1 infected monocyte-derived macrophages, and that Gag induction alters LELs positioning and motility. Further investigation will aim to understand how to target VCC for viral clearance and the design of new approaches to lead us closer to a cure. Supported by CIHR project grants to AJM and RL, NSERC to RL, and ANID-Chile-72210500 to GGC.

HIV-1 hijacks host protein function at multiple steps for its replicative advantage including host proteins involved in membrane trafficking, dynamics and fusion, directed transport, endocytosis, and autophagy. These host proteins have important roles in the replicative cycle of the retrovirus, and they are essential to understand in order to develop effective antiviral strategies. However, despite their potential as possible targets, only a few membrane trafficking proteins have been characterized to serve roles in HIV-1 replication. To elucidate their roles during HIV-1 replication, we performed a CRISPR-Cas9 screen of 140 membrane trafficking proteins in CD4+ TZM-bl reporter cells. This led to the identification of phosphatidylinositol-binding clathrin assembly protein (PICALM) as a meaningful host protein in HIV-1 infection, which was confirmed in subsequent experiments in SUP-T1 CD4+ T cells. The absence of PICALM, a protein that has been previously characterized as intrinsic or related to clathrin-mediated endocytic and autophagy pathways, lead to a reduced viral entry by more than 2-fold (p≤0.001). In addition, PICALM knockout (KO) cells showed changes in other pathways, including the PD-1 and autophagy pathways, resulting in an altered memory phenotype, and changes in intracellular trafficking and abundance of viral HIV-1 Gag. Overall, this work reveals PICALM as a modulator of viral entry, autophagy flux, and immune control of HIV-1 replication.

Background: The vaginal microbiota is a key determinant of HIV susceptibility in cisgender females (cF). In cF, an optimal vaginal microbiota is dominated by Lactobacillus spp., while microbiota dominated by diverse anaerobic bacteria is associated with inflammation and increased risk of acquiring HIV. Estrogen helps support lactobacilli colonization and maintenance, and increased anaerobic diversity is observed post-menopause. Transmasculine individuals (tM) may take testosterone as a component of gender-affirming care. Testosterone therapy reduces serum estrogen levels, but little is known of its effects on the vaginal microenvironment.

Methods: Transmasculine individuals who had been on testosterone for >1 year (n=90) completed online questionnaires and mailed in self-collected vaginal swab samples at 3 time points. Microbiota were characterized through amplification, sequencing, and bioinformatic analyses of the V3-V4 region of the 16S rRNA gene. Microbiota profiles from tM were compared to those from pre- (n=100) and post- (n=100) menopausal cF, previously published by our group.

Results: Lactobacillus spp. were detectable in 73% of tM vaginal samples but were predominant in <10%. Instead, the vaginal microbiota of tM were characterized by diverse communities of anaerobes, including species of Prevotella, Streptococcus, Anaerococcus, Finegoldia, and Peptinophilus, that were distinct from those observed in pre- and post-menopausal cF.

Conclusions: Our findings suggest that the vaginal microbiome of tM has a unique composition and structure, characterized by low abundance of lactobacilli and increased anaerobic diversity. Given the importance of the vaginal microbiota in HIV susceptibility of cF, future work will investigate relationships between microbiota, inflammation, and behavioural practices.

Background: Growth differentiation factor-15 (GDF15) is an atypical member of the TGF-β family involved in tissue reparation, metabolism regulation, and control of appetite. GDF15 levels are one of the best markers of aging, and are elevated upon cardiovascular diseases, some cancers and COVID-19. Herein, we assessed the association between plasma GDF15 levels and clinical characteristics, inflammation and HIV reservoir size in ART-treated PLWH. We analyzed the origin and the effect of GDF15 on immune cells.
Method: Blood was obtained from 55 ART-treated PLWH (median treatment 14.5 years) and 50 uninfected controls. GDF15, markers of inflammation (IL1β, IL6, IL8, TNFα, IP10, CXCL13, sCD14) and non-AIDS comorbidities (soluble urokinase plasminogen activator receptor [suPAR]) were quantified in plasma by ELISA. HIV integrated DNA was quantified by nested-qPCR in sorted CD4 T-cells. GDF15 producing cells were identified by flow cytometry. GDF15 response was analyzed by flow cytometry.
Results: Plasma GDF15 levels were higher in PLWH than uninfected controls (p<0.001). GDF15 levels were not associated with inflammation, nor gut permeability markers, and was not induced upon in vitro inflammatory stimulations. Conversely to other markers, GDF15 levels were strongly associated with integrated HIV DNA levels (r=0.59, p<0.01) and suPAR (0.68, p<0.0001) independently of age, sex, and CD4 count. Ex vivo, GDF15 protein was only found in monocytes, and not in T or B cells, NK cells nor dendritic cells in PBMC. In vitro, stimulation with recombinant GDF15 induced tyrosine phosphorylation in CD4 and CD8 T-cells, indicating a potential direct effect of GDF15 on those cells. GFRAL, the only currently known receptor of GDF15, was not expressed on T-cells.
Conclusion: As classical markers of inflammation were not linked with GDF15 levels, our results suggest a new link, possibly metabolic, that might be targeted to decrease HIV persistence and risk of non-AIDS comorbidities in ART-treated PLWH.

Viruses are obligate intracellular pathogens that require many host cell components to complete their life cycles. Several genome-wide screens have been performed across multiple viral models that have identified hundreds of these viral host dependency factors (HDFs). These HDFs may be good candidates to develop novel host-directed antivirals. However, defining which HDF may make good targets and which HDF may lead to drug toxicity is challenging. Intersecting genes found to be viral HDFs and also not essential for host function, may open new avenues for the development of therapies. We performed a comprehensive literature review and identified 27 studies covering HIV, Hepatitis C, Hepatitis D, SARS-CoV-2, SARS-CoV, Ebola, Influenza A, Zika, Dengue and West Nile viral models. A cumulative total of 3248 HDFs were collected and 320 genes were implicated in more than 1 virus. With software like DAVIDv6.8 and REACTOME, we determined that the most hijacked biological pathway by all these viruses is phagosome acidification (mostly used for cell entry). These HDFs were then intersected with the genome aggregation database (gnomAD) using an in-house bioinformatic pipeline, the genome non-essentiality and loss-of-function identifier (gNALI). The gnomAD resource contains >125,000 human exome and >15,000 whole-genome sequences from healthy individuals with specific information on genes harbouring loss-of-function polymorphisms. The output of gNALI is a list of genes that are HDFs with observed loss-of-function polymorphisms from gnomAD. Finally, we narrowed down six genes that can be potentially targeted as host-directed antiviral targets for HIV and one other virus for our future wet-lab study.

Molecular dating can provide insights into the timing and within-host dynamics of viral infection. Leading methods for molecular dating involve Bayesian analysis, where prior knowledge informs estimates of unknown dates. Estimating the time of the most recent common ancestor (tMRCA) of HIV sequences isolated from a person living with HIV (PLHIV) can help us estimate the timing of infection. Furthermore, estimating the integration dates of proviral sequences sampled during long-term suppressive antiretroviral therapy (ART) can help reveal the dynamics of the persistent reservoir, thereby assisting HIV cure investigations. We present a Java package, BBD, that extends BEAST2’s functionality to perform date estimation with Bayesian methods. We use our package to estimate infection and proviral integration dates in simulated and empirical data sets of longitudinally sampled HIV sequences to determine which priors on the dates produce the most accurate results. We compared four different priors on the tMRCA, as well as no prior, to estimate infection dates using published longitudinal HIV sequence data sets; these were collected prior to ART initiation from twelve PLHIVs who recorded a negative HIV test within 29-116 days of their first positive HIV test. We compared nine different priors to estimate proviral integration dates of HIV sequences from the persistent reservoir using simulated and published empirical data sets of longitudinal HIV sequences collected prior to, and following initiation of suppressive ART. We found marked differences in the estimated infection and proviral integration dates using the different priors, and without using a prior. In particular, when no prior was specified, the tMRCA of an individual tended to precede their last negative HIV test. Specifying appropriate priors provides more accurate and robust results in the date estimation of HIV. Precise understanding of HIV dynamics will aid in our efforts for HIV control and cure.

Background: Globally 1.5 million new HIV infections occurred in 2020, therefore, new prevention methods are needed. Inflammation is a risk factor for HIV acquisition as it attracts HIV target cells to the female genital tract (FGT). Our lab conducted a study to reduce FGT HIV target cells using safe, affordable, and globally available anti-inflammatory: acetylsalicylic acid (ASA/Aspirin). We found ASA decreased the proportion of FGT HIV target cells (CD4+CDCR5+Tcells) by 35%. However, the mechanism remains unknown.
Goal: To assess if ASA use reduces mediators of inflammatory pathways such as the lipoxygenase pathway.
Methods: Women from Nairobi, Kenya took low dose ASA (81mg) daily for 6 weeks. Blood was drawn at baseline and following 6 weeks daily ASA. Plasma was frozen at -80˚C and shipped to Winnipeg, Canada. Oxylipins in the plasma were stabilized with antioxidants, spiked with an internal standard, extracted on Strata-X-SPE columns and quantified using liquid chromatography mass spectroscopy. Oxylipins from 12 possible pathways were assessed.
Results: All detected cyclooxygenase metabolites from arachidonic acid were downregulated, 4/6 statistically significantly so. We detected at least one metabolite from 9 of the 12 possible oxylipin pathways, 4 pathways had more than one analyte significantly different following 6 weeks ASA. Interestingly, the majority of lipoxygenase metabolites from all 4 pathways were significantly downregulated: 5/13 from arachidonic acid, 2/10 from docosahexaenoic acid, 2/6 linoleic acid, and 2/2 from dihomo-γ-linoleic acid.
Conclusion/Discussion: We show that the following 6 weeks of ASA treatment, metabolites from both the lipoxygenase and cyclooxygenase pathways were down regulated. While ASA directly inhibits cyclooxygenase function, this is not the case for lipoxygenase. However, inflammation increases lipoxygenase expression and ASA reduced inflammation in our cohort. We speculate that ASA-associated reduction in inflammation, decreased lipoxygenase expression resulting in decreased lipoxygenase metabolites.

In silico methods for epitope prediction have become a critical part of vaccine and therapeutic design during the COVID-19 pandemic, but intra-species comparison of putative epitopes remains a difficult task that is laborious and prone to human error. Created for studying SARS-CoV-2 variants of concern, Comparative Analysis of Variant Epitope Sequences (CAVES) is a novel tool designed for rapid comparative analyses of epitopes amongst closely related pathogens, substantially reducing the required time and user workload.

CAVES utilizes two comparison levels to extract information about the relationship between epitopes from two sequences and their relevance in published literature. The level-one analysis compares two epitope prediction files from the Immune Epitope Database-Analysis Resource (IEDB-AR), while the level-two analysis incorporates search results from the IEDB database of experimentally confirmed epitopes, effectively characterizing the similarities/differences amongst putative epitopes and determining whether they exist in the current database.

CAVES utilizes a graphical user interface on Windows operating systems, making it widely accessible regardless of coding expertise. Each comparison level sorts epitopes into categories of exact matches, partial matches, or novel epitopes by the degree to which they match with peptides from the opposing file. Furthermore, CAVES uses sequence positional data to improve its accuracy and speed, taking only a fraction of the time required by manual analyses and removing the risk of human error.

CAVES is highly applicable for evolutionary analyses as epitopes labelled as exact matches can be considered as conserved amongst the two sequences, while those labelled as novel are unique epitopes that were gained/lost during pathogen evolution. CAVES can be used to determine the similarities/differences of epitopes amongst any species and is particularly relevant for pathogens with high mutability like HIV-1. Additionally, CAVES is widely applicable beyond evolutionary objectives and can be used for confirmatory purposes when using multiple epitope prediction programs.

Efforts to develop a practical HIV-1 cure have largely focused on immune-directed strategies which rely on the action of cytotoxic T lymphocytes (CTLs) to clear infected cells in vivo. Yet, as HIV-1-infected cells are intrinsically resistant to CTL killing, these cures exhibit poor efficacy. To evade CTL killing, HIV-1 encodes the protein, Nef, which blocks viral antigen presentation by binding and activating a suite of Src family kinases (SFKs) that ultimately leads to the downregulation of cell surface major histocompatibility complex class I (MHC-I). Inhibiting the Nef–SFK interaction may therefore be a direct approach to functionalize an immune-directed HIV-1 cure by rescuing cell surface MHC-I and enhancing CTL-mediated infected cell killing. Previously, our group identified a dipeptide derivative predicted to bind Nef, termed H3-1. Subsequent studies established H3-1 to inhibit the Nef–SFK interaction in vitro and to modestly rescue cell surface MHC-I in cell culture, while mouse studies identified H3-1 to be rapidly cleared in vivo. Therefore, we aim to use H3-1 as a chemical starting point to develop Nef–SFK interaction inhibitors with improved in vivo stability. We hypothesize that H3-1’s peptidic structure is central to its instability in vivo and thus are using organic synthesis to generate a peptidomimetic panel of H3-1 analogues. Specifically, we are pursuing the thioamide and methyleneamino replacements of H3-1’s amide group, and the methyl ester, amide and tetrazole replacements of H3-1’s carboxylic acid group. In parallel, using a series of cell culture experiments, we are characterizing each analog on variables including toxicity, Nef–SFK interaction inhibition, MHC-I rescue and biostability. By integrating each of these measures, we aim to identify a next-generation in vivo stable Nef–SFK inhibitor, which will enable us to evaluate the utility of Nef-mediated MHC-I downregulation blockade in an HIV-1 cure.

Background: Dolutegravir (DTG), an integrase strand transfer inhibitor (INSTI), is a WHO-alternative first-line regimen. Initial findings from an observational study in Botswana showed an elevated incidence of neural tube defects (NTDs) with peri-conceptional exposure to DTG. We have previously shown that DTG exposure yielding therapeutic levels in pregnant mice on a folate sufficient diet was associated with higher rates of fetal anomalies compared to control-treated mice. Here we explore potential DTG reproductive toxicities in a folate deficient pregnancy mouse model.

Methods: Female C57BL/6 mice fed a folic acid deficient diet for a minimum of 2 weeks, were mated and randomly allocated to either control (water) or 1x-DTG (2.5mg/kg DTG+50mg/kg tenofovir 33.3mg/kg emtricitabine). Drug/water was administered once daily by oral gavage from day of plug detection to sacrifice at E15.5. Fetuses were assessed for anomalies by two independent reviewers who were blinded to treatment allocation. Mixed effects logistic regression was used to assess differences between treatment groups accounting for litter effects.

Results: A total of 1533 fetuses from 209 litters were assessed (control n=103 litters, 756 fetuses; 1x-DTG n=106 litters, 777 fetuses). Percent viability, placental weight, fetal weight, fetal/placenta weight ratio, and maternal weight gain did not differ between groups. Crown-lump length was lower and head width was higher in the 1x-DTG vs. control groups. Seven NTDs (exencephaly, n=2; encephalocele, n=3; spinal bifida, n=2) were observed in the 1x-DTG group (7/777=0.9%), with no NTDs in controls. Fetuses exposed to 1x-DTG also had higher rates of severe turning defects (2.2% vs. 0.4%, p=0.04), abdominal wall defects (3.5% vs. 0.4%, p=0.04), limb defects (3.9% vs. 0.5%, p=0.001), cranial/spinal bleeds (15.7% vs. 5.4%, p<0.001), and severe edema (7.0% vs. 1.3%, p<0.001).

Conclusion: DTG treatment was associated with higher rates of fetal anomalies compared to controls in pregnant mice on a folate-deficient diet.

Background: Targeting the latently infected CD4+ T-cells is one of the main challenges in finding a cure for HIV. Although these cells cannot be phenotypically distinguished from their uninfected counterparts, we have demonstrated that the cells latently/persistently infected with HIV have impaired interferon signaling. This defect makes them susceptible to be selectively infected and killed by the oncolytic virus (OV) MG1. Both increasing its specificity to infected cells and sensitizing these cells to its killing can be expected to make MG1 a more effective therapeutic. This project aims to pseudotype MG1 with HIV envelope protein gp160, to enhance targeting of infected cells, and determine if combining MG1 with the SMAC mimetics (SM) LCL-161 and AEG-40730 can increase killing of cells that are latently infected with HIV.
Methodology: For pseudotyping experiments, restriction enzyme cloning, and Gibson Assembly were used to create full length and modified gp160 containing MG1 clones. For SM and MG1 combination experiments, Jurkat cells and their latently HIV infected counterpart J1.1 cells were infected with MG1 and treated with the SM LCL-161 and AEG-40730, and their viability and infection percentage were assessed 24h and 48h post treatment via flow cytometry.
Results: MG1 clones have been verified by sequence analysis and are in the process of being rescued by transfection. The divalent SM AEG-40730 has greater killing effect compared to the monovalent LCL-161 in both cell lines, but neither SM show a preferential killing of J1.1 cells compared to Jurkat cells. Combination of a low dose of LCL-161 and MG1 infection shows significantly increased killing of J1.1 cells.
Conclusion: Both OV and SM therapy are promising therapeutic agents in the field of HIV. A combination of OV and SM therapy can potentially be a new strategy to eliminate cells latently infected with HIV.

Older adults remain at higher risk of severe COVID-19. Two-dose mRNA vaccines reduce hospitalization and mortality, but immune protection declines, and third doses are now recommended. We examined immune durability up to 6 months after two vaccine doses, and immunogenicity after a third vaccine dose in 151 adults ranging in age from 24 to 98 years.

Specimens were collected from 81 healthcare workers (median age 41 years), 56 older adults (median 78 years) and 14 COVID-19 convalescent individuals (median 48 years), at one, three and six months following the second dose and from 15 HCW, 28 older adults and 3 convalescent individuals at one month following a third dose. Binding antibodies to the SARS-CoV-2 spike receptor binding domain were quantified using an immunoassay (Roche Elecsys). Virus neutralizing activity was assessed using a live SARS-CoV-2 infection assay.

Compared to healthcare workers, older adults displayed ~1.9-fold lower peak binding antibodies one month after the second dose (p<0.001) and modestly faster rates of antibody decline thereafter (p=0.005). Peak neutralizing activity was 4-fold lower in older adults one month after the second dose (p<0.001) and became undetectable in most individuals by six months. One month after a third dose, binding antibodies and neutralizing activities surpassed peak values after two doses in both groups, and differences were no longer statistically significant. Compared to both naïve groups, convalescents displayed slower rates of binding antibody decline (p<0.006) and maintained higher neutralizing activity six months after the second dose.

Immune responses to two-dose COVID-19 mRNA vaccines are attenuated in older adults, but binding antibodies and neutralizing activity are enhanced significantly by a third dose. Potentially faster rates of antibody decline in older adults suggest that waning of responses should be monitored for this group.

Introduction: Oxytocin, a neuropeptide released during sexual activity and physical touch, is shown to have anti-inflammatory effects on gut and skin epithelium. Human immunodeficiency virus (HIV) takes advantage of inflammation at the female genital tract (FGT) to facilitate its replication and transmission. This study examines if oxytocin reduces immune activation, improves epithelial wound healing, and protects HIV target cells from infection at the FGT.
Methods: Vaginal (Vk2), ectocervical (Ect1), and endocervical (End1) cell lines were grown in monolayers. RNA isolated from these cell lines, before and after stimulation with 1 µg poly(I:C)/LyoVec and/or 10, 100, 1000, or 10 000 pg/mL oxytocin, was evaluated for the effect of oxytocin on inflammatory gene expression using RT-qPCR. Oxytocin’s effects on wound healing were assessed using scratch assays with monolayers pre-treated with 10 000 pg/mL oxytocin. Triple-knockout mice, transplanted with stem cells from human bone marrow and fetal liver and thymus (TKO-hBLT), were used as an in vivo model to assess whether intravaginal administration of oxytocin (1.6 IU/kg) directly impacts immune activation of peripheral blood mononuclear cells (PBMCs) by measuring HLA-DR, CD38, CD69, and CCR5 expression via flow cytometry.
Results: Oxytocin (10 000 pg/mL) treatment reduced the expression of both IL-6 and IL-1β in End1 cells by 50% (p<0.05; n=6). This was reversed by poly(I:C)/LyoVec stimulation. Scratch assays showed no effect of oxytocin on wound closure. Data from TKO-hBLT mice showed that intravaginal oxytocin had no effect on the expression of PBMC activation markers (n=4).
Discussion: Oxytocin reduced the expression of inflammatory genes in End1 cells, which implies its ability to modulate immune activation at the vulnerable endocervical site. Although oxytocin had no direct effect on FGT epithelial wound healing, its indirect role in epithelial barrier properties should be investigated. Further investigation of oxytocin’s role in regulating immune activation at FGT mucosa is needed.

Closed Env is resistant to most binding antibodies because immune cells have limited access to CD4-induced conserved epitopes buried inside the closed Env. We previously reported that HIV-1 containing N425K mutation had a higher binding affinity for CD4 than its wildtype due to the generation of a new hydrogen bond and a pi-pi stacking, both of which lock Env with CD4. K425, located at the β20 bridging sheet, forms the gp120 Phe43 pocket, which is a key regulator to initiate CD4-induced conformational changes on both gp120 and gp41 subunits. Despite the increased infectivity and binding affinity of HIV-1 containing K425, K425 is rarely seen in naturally infected individuals. We hypothesized HIV-K425 may stabilize CD4-bound structures and expose inner conserved epitopes upon CD4 contact, thus this mutant can be rapidly eliminated. Therefore, Env-K425 could be a great candidate as an immunogen for vaccine development. We also compared the efficacy of multiple vaccine vectors, including virus-like particle (VLP), proviral DNA and vesicular stomatitis virus (VSV)-vectored HIV-1 vaccines, carrying the same immunogens, HIV-1A74_gp120 containing K425 or N425, using mouse and macaque models. In BALB-c mice, VLPs, compared with the DNA vaccines, significantly increased the number of HIV-1-specific central memory T cells, gp120 binding antibodies and neutralizing antibodies. Interestingly, mouse splenocytes after being treated with recall antigen generated a novel CD4/CD8 double-positive (DP) T cell population that was rare in media negative or PMA positive controls. Moreover, the percentage of central memory subset in DP T cells was higher than that of CD4/CD8 single-positive T cells. Based on the FDA-approved VSV-vectored EBOV vaccine, we engineered replicating a VSV-vectored HIV vaccine co-expressing HIV-1_gp120-K425 and Ebola-GP, and it showed 67% protection against SHIV in macaques. In summary, HIV vaccines presenting CD4-bound Env may be a solution to elicit potent immune responses targeting inner conserved epitopes.

It is estimated that over 37.5 million people are living with HIV worldwide, over 1.5 million new infections were reported and 680,000 AIDS-related deaths were recorded in 2020. With the growing number of new HIV infection and AIDS related deaths worldwide, there is an urgent need for new and effective means to tackle the spread of HIV infection. In our project, we aim to develop a 3D printed intravaginal scaffold as a two-pronged approach to the problem. Acetylsalicylic acid (ASA), an anti-inflammatory drug, will be delivered along with tenofovir disoproxil fumarate (TDF) to help reduce vaginal inflammation and enhance the anti-viral activity. 3D printing will be employed to design and fabricate the scaffold system to regulate drug release over an extended period thereby improving adherence among patients. These macaque sized intravaginal rings will be printed using fused filament fabrication type of 3D printing and thermoplastic polyurethane 60D35 polymer filament. ASA and TDF nanoparticles will be suspended in a gel matrix and loaded onto the intravaginal rings (IVR) for a controlled drug release. Ring design parameters will be optimized to achieve an extended release of the drugs. The nanoparticles prepared had an average diameter of 187±1.73nm and a zeta potential of -29.3±1.63mV. The double emulsion technique of nanoparticle fabrication achieved an encapsulation efficiency of 84.65±0.84%. A cyclic oligosaccharide, β-Cyclodextrin and non-aqueous gel formulations were tried to improve the stability of acetylsalicylic acid, an ester. Aspirin loaded hydroxyethyl cellulose based non aqueous gel showed the highest stability and temperature was found to be inversely proportional to the stability. Cytotoxicity studies on SupT1 cell lines were performed using ASA and TDF and found to be safe at practical concentrations. Future studies involve studying the targeting potential of nanoparticles and optimization of the final IVR for an extended release.

Interferon-alpha (IFN-α) has been associated with excessive immune activation and dysfunction during HIV-1 infection. However, evidence suggests specific IFN-α subtypes may be beneficial rather than detrimental. Our previous work showed that IFN-α subtypes differentially control HIV-1 infection and mediate distinct effects on immune function. Clinical use of the IFN-α2 subtype has not been highly effective in reducing viral or proviral HIV-1 and high levels of endogenous IFN-α2 have been associated with CD8+ T cell hyperactivation and dysfunction in HIV-1 patients. Our previous study with the IFN-α14 subtype suggested that some IFN-α subtypes may be beneficial during HIV-1 infection. This study compared the effects of treatment with two different IFN-α subtypes on indicators of T-cell activation and dysfunction during HIV-1 infection. Using HIV-1-infected TKO-BLT humanized mice, we demonstrated that after 3 weeks of treatment, IFN-α14 had significantly reduced viremia and markers of CD8+ T cell-related dysfunction such as hyperactivation, exhaustion and apoptosis comparable to healthy controls and unlike anti-retroviral therapy (ART), low levels of these markers were maintained even after the treatment was withdrawn. Mice treated with IFN-α14 had a greater Tnaïve/Teffector memory ratio of CD8+ T cell profile as opposed to the development of the larger effector memory subset observed in HIV-1 infected and IFN-α2 treated mice. Although IFN-α14 treatment reduced the activation profile and proliferative capacity of CD8+ T cells, it did not change their ability to secrete cytokines or degranulate upon stimulation ex vivo. In addition, IFN-α14 treatment did not alter the CD4+ T cell count supporting the hypothesis that IFN-α14 treatment does not exacerbate HIV-1 disease progression and may have therapeutic potential to alleviate CD8+ T cell dysfunction during HIV-1 infection.

This work was supported by Saskatchewan Health Research Foundation (SHRF) Canada

HIV set point viral load (spVL) is a predictor of HIV disease progression and transmission, thus restriction of spVL is key to ending the AIDS pandemic. There is variability in HIV spVL among individuals, with host genetics, particularly in the HLA region, contributing to this variability. However, HIV can develop escape mutations to evade host pressure, counteracting their effect. A recent GWAS of >3,800 HIV+ individuals of African ancestry, performed by our group, identified a novel locus on chromosome 1 that associates with control of HIV replication. A variant within this region, rs59784663(G), is associated with an average ~0.3 log10 reduction in HIV spVL (p=2.0x10-9) and is downstream from the protein coding gene chromodomain helicase DNA binding protein 1 like (CHD1L). Despite the spVL decreasing effect of rs59784663, some individuals with the protective allele still have high spVLs. Given the high mutation rate and short generation time of HIV, we hypothesized that selective mutation of the viral genome would allow HIV to escape restriction by CHD1L. We test this hypothesis by conducting a viral genome-to-host genome analysis in 97 individuals from South Africa with both human and viral genomic data available. Human genetic variants in the CHD1L region and HLA are tested for association with amino acid (AA) variants in the HIV proteome. Analyses found a significant association between the CHD1L variant rs59784663 and AA codon 248 of HIV reverse transcriptase (p=9.9x10-3). In addition, there were significant associations between HLA B*81 (p=1.5x10-5) and HLA C*18 (p=1.4x10-3) with AA codon 4 and HLAB*58 with AA codon 196 (p=1.0x10-3) in HIV reverse transcriptase. Ongoing work will seek to replicate these associations in other African populations. This study has the potential to reveal regions of conflict between the host and viral genomes, increasing our understanding of viral evolution and host control of HIV.

Background: Lung remains an understudied site of HIV persistence. We characterized proviral diversity and compartmentalization in blood and broncheoalveolar lavage (BAL) in individuals with HIV on long-term cART.

Methods: Subgenomic HIV sequences (nef region) were characterized by single-genome approaches from matched blood (buffy coat) and BAL specimens from 9 individuals with suppressed viral loads on cART. Markov chain Monte Carlo methods were used to infer a distribution of ~1,000 within-host phylogenies in MrBayes. Diversity metrics were calculated from the highest-likelihood phylogeny. Genetic compartmentalization was assessed using Hudson, Boos and Kaplan's nonparametric test for population structure (KST) and the Slatkin-Maddison (SM) test, with the latter results conditioned over all trees.

Results: We isolated 1,025 nef sequences (788 blood, 237 BAL). Of these, 882 (86%) were intact (i.e., non-hypermutated, without other defects), yielding medians of 78 (Q1-Q3=58-90) and 14 (Q1-Q3=6-37) intact sequences/individual for blood and BAL, respectively. Consistent with clonal expansion, 331/882 intact sequences (38%) were identical to another collected, where 7/9 individuals harbored at least one specific sequence that was present in both blood and BAL. In three participants, identical sequences represented ≥50% of their overall pool, whereas in others, most sequences were unique. Overall, the diversity of unique HIV sequences in blood reflected that in BAL (Spearman’s r=0.75, p=0.02). When considering unique sequences per compartment, strong evidence for compartmentalization, defined as statistically significant support in both KST and SM test results, were not observed for any participant's dataset.

Conclusion: Proviral nef sequence diversity in blood during cART is representative of that in the lung, with no evidence of genetic compartmentalization between these two sites. The presence of identical HIV sequences in blood and lung is consistent with migration of clonally-expanded reservoir cells between these two sites. Eradication strategies will need to contend with genetically diverse HIV in blood and tissues.

Short-chain fatty acids (SCFAs) are microbial-derived metabolites that enter the circulation. SCFAs are reported to maintain the gut mucosal barrier and regulate the inflammation of gut mucosa. Paradoxically, at vaginal mucosa, high level of SCFAs is associated with bacterial vaginosis (BV) and vaginal inflammation. Since the cervicovaginal epithelial barrier function and vaginal mucosal inflammation are critical for the susceptibility to HIV and HPV infection, this study focuses on deciphering the relationship between vaginal SCFAs and inflammatory cytokine/chemokine levels in cervicovaginal fluid (CVF) and defining the direct effects of SCFAs on cervicovaginal epithelial integrity.
The CVF samples from healthy (n=27) and HPV-infected non-menopausal women with low-grade squamous intraepithelial lesions (LSIL) (n=14) were analyzed for SCFAs and 18 inflammatory cytokine/chemokine. HPV DNA in the vaginal samples was detected by PCR.
Vaginal SCFAs detected were acetate (0.16-2.8mM), butyrate (0.35-41.9µM), propionate (0.23-67.0µM), beta-hydroxybutyrate (0.1-43.4µM), and valerate (0.02-0.6µM). Unexpectedly, the SCFAs levels in LSIL-HPV CVF were not different from the controls. We further found that levels of vaginal SCFAs were significantly higher than plasma SCFAs (p-values <0.01). Furthermore, the LSIL-HPV CVF and the HPV-negative control CVF had similar levels of pro-inflammatory cytokine/chemokine. In vitro study showed that treatment of polarized, multilayered cervicovaginal epithelium with sodium butyrate (5mM) or propionate (5mM) for 48 hours resulted in increased epithelial permeability, with reduced trans-epithelial electrical resistance across the epithelium.
Taken together, SCFA levels are similar in the CVF of the LSIL-HPV and the HPV negative participants. It’s perhaps, due to the similar status of low vaginal inflammation in both groups of participants; it agrees with the similar levels of cytokine/chemokine in the CVF from both groups. We provide evidence that high level of SCFAs could directly impair the cervical-vaginal epithelial barrier integrity. Work is ongoing to validate this observation in the primary epithelium.

Interferon-induced transmembrane (IFITM) proteins have been shown to inhibit a wide range of enveloped viruses including influenza virus, Dengue virus, and human immunodeficiency virus type 1 (HIV-1). The key role of IFITM3 protein in host antiviral defense is supported by the severe morbidity and high mortality in IFITM3 knockout mice upon influenza virus infection, as well as severe influenza virus infection and rapid progress of HIV-1 infection associated with single nucleotide polymorphisms in IFITM3. IFITM3 restricts virus infection by impeding virus entry. The underlying molecular mechanisms include the alteration of membrane properties by IFITM3, such as membrane fluidity and curvature, which halts the fusion of viral membrane with cellular membrane. It is incompletely understood how IFITM3 protein changes membrane properties. One hypothesis is that IFITM3 is localized to lipid rafts partly because of its palmitoylation at the cysteine residues, thus increasing membrane rigidity. However, a recent study showed partition of IFITM3 to the non-raft region in lipid vesicles. To determine the distribution of IFITM3 on the plasma membrane of intact cells with reference to lipid rafts, we used the single-molecule imaging technology that allows the direct observation of IFITM3 organization at a 10 nm resolution, which is 10 times smaller than an HIV-1 particle. Both the ectopically expressed IFITM3 and the interferon-induced endogenous IFITM3 form clusters of 50 to 100 nm in size, and are interspersed between lipid rafts that are labeled with cholera toxin B. Our data suggest that IFITM3 increases the rigidity of membranes by acting on the non-raft, disordered regions.

Introduction: Even with sustained antiretroviral therapy (ART)-mediated virologic suppression, HIV is associated with some persistent inflammation, increasing risk of comorbidities. The 2-drug regimen dolutegravir (DTG) plus lamivudine (3TC) has demonstrated durable, non-inferior efficacy compared with 3- and 4-drug regimens (3/4DRs) in ART-naive and ART-experienced people with HIV-1 (PWH).
Objectives: This systematic review summarized randomized controlled trial (RCT) and real-world evidence evaluating inflammatory and atherogenesis biomarkers with DTG + 3TC in ART-experienced PWH.
Materials and Methods: Ovid MEDLINE®, Embase®, PubMed, and Cochrane library databases were searched for studies published from January 1, 2013, to July 14, 2021. Relevant data from the 2021 International AIDS Society (IAS) Conference on HIV Science and IDWeek™ 2021 were also retrieved. Eligible studies included real-world evidence and RCTs evaluating switch to DTG + 3TC in ART-experienced PWH aged ≥18 years with data on CD4+/CD8+ ratio or inflammatory and atherogenesis biomarkers.
Results: Overall, 4 publications representing 2 RCTs (DTG/3TC: SALSA, n=246; TANGO, n=369) and 6 publications of real-world evidence (DTG + 3TC: N=1000) were included. Across RCTs, no consistent pattern of change in biomarkers was observed between DTG/3TC and 3/4DR comparators, except for reduced soluble CD14 (favored DTG/3TC in SALSA at Week 48 and TANGO at Weeks 48 and 144; P<0.05) and IL-6 (favored TAF-based regimens in TANGO at Weeks 48 and 144; P<0.05). In the one real-world study evaluating changes in inflammatory biomarkers (N=67), median soluble CD14 levels significantly decreased at Week 48 post-DTG + 3TC switch (P<0.001). Levels of other biomarkers (including IL-6) remained stable. In all 6 real-world studies, CD4+/CD8+ ratio increased post-switch to DTG + 3TC (follow-up, 12-60 months).
Conclusion: Results showed no consistent impact on inflammatory and atherogenesis biomarkers post-switch to DTG + 3TC vs 3/4DR comparators, suggesting no evidence of an impact on inflammation after switching from a 3/4DR to DTG/3TC.

Despite African populations having the largest burden of HIV disease, the majority of large-scale HIV host genetic studies have been in European populations. To address this gap, our lab conducted a genome-wide association study of HIV set-point viral load (spVL), a strong indicator of disease progression with a known host genetic component, in >3,800 individuals of African ancestry. We detected a novel region of chromosome 1 significantly associated with decreased HIV spVL. Variants in high linkage disequilibrium (R2 ≥ 0.6) with the top SNP, rs59784663 (p= 6.4E-10, β = -0.3), overlap three coding genes: CHD1L, PRKAB2, and FMO5, none of which have been previously implicated in HIV pathogenesis. Statistical fine-mapping of the chromosome 1 region revealed that two non-coding variants, rs73004025 and rs7519713, provide a significantly better model than the top SNP alone (two-way ANOVA, p < 0.01) and functional assessment suggests these variants may impact gene expression. To test for differences in gene expression, cryopreserved PBMCs from HIV+ individuals (N=24) with different genotypes at the top associated SNP were sorted into CD4+ T cell and monocyte fractions. The CD4+ T cell fraction was divided into an unstimulated and stimulated (CD3/CD28 for 8 hours) fraction before RNA collection. Due to low cell numbers, RNA was collected only from unstimulated monocytes. Gene expression of CHD1L, PRKAB2, and FMO5 was measured using Taqman RT-qPCR assays with TBP, RPL13A, and SDHA as CD4+ T cell housekeeping genes, and in monocytes, HPRT1 and SDHA. We observed no significant differences or trends in expression of PRKAB2 or FMO5, but a trend towards lower CHD1L expression in individuals carrying the variant allele in stimulated CD4+ T cells and unstimulated monocytes. Ongoing work will increase the sample size of individuals carrying the reference and alternate alleles to increase the power of detecting significant differences.

Purpose: Microbicides are an excellent alternative to condoms to help reduce transmission of human immunodeficiency virus (HIV). An intravaginal ring (IVR) would be a suitable platform that can provide controlled delivery of drugs within the female genital tract with high patient acceptance. We propose to develop a segmented combination IVR whereby one-half of the IVR will be loaded with hydroxychloroquine (HCQ), an immuno-modulatory drug that can induce a quiescent state in T cells and the other half will be coated with a pH-responsive film for the rapid release of small interfering RNA (siRNA)-encapsulated nanoparticles (siRNA-NP) against CCR5 gene, triggered by an increase in vaginal pH due to the presence of seminal fluid.

Methods: Solid lipid nanoparticles will be used to encapsulate siRNA. siRNA-NP will be mixed with Eudragit L100 and used to coat a polyurethane IVR segment fabricated by hot-melt injection molding. HCQ will be loaded in a reservoir-type polyurethane IVR segment. Release studies will be performed and cytotoxicity of the IVR segments will be evaluated on vaginal cell lines and vaginal flora. The expression of immune activation markers such as CD69 and HLA-DR will be accessed, and the CCR5 gene expression level.

Results: IVR segments coated with a pH-sensitive polymer released negligible amounts of fluorescent NP at pH 4.2. Once the pH was increased to pH8.2, there was a rapid release of 12% within 4 hrs. The reservoir-type IVR segment containing HCQ continuously released drug up to 21 days with a near zero-order release profile (R2 value =0.99) with a mean daily release of 17.013.6 g/mL. Cytotoxicity evaluation of IVR segments on vaginal cells and lactobacilli demonstrated no significant changes in cell viability or growth, respectively. The relative gene expression of CCR5 in cells treated with the siRNA-NP was significantly reduced by 83 ± 5.1% compared to non-treated cells.

Identifying novel biomarkers early during HIV-1 infection may provide valuable insight into a patient’s prognosis and treatment options, especially in geographical areas with low resources. Cytokines, as well as chemokines, define classes of proteins responsible for local and systemic signaling between cell populations of the immune system. During viral infections, cytokines recruit and activate immune cell defense mechanisms against invading pathogens. Upon HIV-1 infection, the virus can enter in the endocervical tract. At this site, it is unknown which cytokines are secreted to mount an immune response and subsequently attract monocytes. Using Luminex MagPix assays, we examined endocervical cytokine profiles obtained from cervical swabs of HIV-infected women (n = 20) from the Uganda GS Cohort. This longitudinal cohort of HIV-1 infected Ugandan and Zimbabwean women includes individuals infected with HIV-1 subtypes A, C, and D and follows the timeline of disease progression from infection to CD4+ T cell decline, requiring the administration of antiretroviral therapy following World Health Organization guidelines.We correlated cytokine levels to the viral load within the endocervical tract at a time point below 60 days post infection. Of the 14 cytokines measured: interferon gamma, monocyte chemoattractant protein 1, macrophage inflammatory protein and C-X-C motif chemokine 10 positively correlated to viral load within the endocervical tract. Interestingly, these cytokines have clinical significance as they are involved in inflammation, macrophage recruitment and response to viral infection. Overall, our results demonstrate a relationship between HIV disease progression with early endocervical cytokine profiles involved in pro-inflammatory and chemotactic responses.

Background: Unwanted pregnancies and sexually transmitted infections (STIs) are major health concerns of women worldwide. These concerns have prompted efforts to develop Multipurpose Prevention Technologies (MPTs), which simultaneously provide contraception and prevent STIs, including HIV. LL-37, an effective spermicide on human sperm, has broad antimicrobial activity including in vitro activity against HIV. 17BIPHE2 is a truncated LL-37 peptide, engineered to contain 5 unnatural residues, thus limiting its protease degradation by vaginal fluid. Hence, this AMP represents a promising MPT agent.

Methods: PMA-stimulated ACH-2 cells, a chronically HIV-infected T cell line, were incubated with LL-37 or 17BIPHE2, and HIV replication was evaluated by p24 concentration in the supernatant via ELISA. In addition, HIV was incubated with 17BIPHE2 prior to infection of various target cells for HIV infection. Alternatively, target cells were incubated with 17BIPHE2 prior to HIV infection. Infection was quantified by luciferase activity in an HIV reporter TZM-bl cell line and by p24 ELISA in activated PBMC and CD4+ T cells.

Results: In ACH-2 cells, there was significant reduction in p24 production when cells were treated with 17BIPHE2, but not LL-37. When 17BIPHE2 was pre-incubated with HIV prior to infection and present during infection, viral replication decreased in the TZM-bl reporter cell line, but this result was not recapitulated in the primary activated cells, PBMCs nor isolated CD4+ T cells. Conversely, pre-incubation of 17BIPHE2 with target cells prior to infection significantly inhibited HIV infection in a dose-dependent manner. Initial mechanistic studies involving evaluation of cell-surface markers of activation and co-receptor expression indicated no change between untreated and 17BIPHE2-treated cells.

Conclusion: 17BIPHE2 may act on the cell or on the cell/virus interaction rather than on the virus itself to inhibit HIV infection and presents a promising anti-HIV therapy that may be developed into an effective MPT.

Human myxovirus resistance protein B (MxB, also known as Mx2) is a dynamin-like GTPase. Its expression is inducible by type I interferon. Multiple RNA viruses, including human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus, and DNA viruses such as herpesviruses and hepatitis B virus have been shown to be restricted by MxB. MxB exhibits a signature localization to the nuclear pore complex (NPC) on the nuclear envelope, which is associated with its antiviral function. Yet, it remains unclear how MxB is anchored to the NPC. During mitosis, with nuclear envelope breakdown, NPC also dissociates, and many of its components are distributed to various subcellular locations. We hypothesize that monitoring the distribution of MxB during mitosis by confocal imaging may provide insights on which NPC components MxB associates with. To test this, we employed the CRISPR-Cas9 technology to generate MxB-RFP knock-in HeLa cell clones which can be used to track the distribution of endogenous MxB during mitosis by live-cell imaging. At interphase, MxB-RFP was observed at the nuclear envelope. When cells divide and the nuclear envelope breaks down, MxB forms small granules instead of complete dispersal. At metaphase, when chromosomes are aligned at the equatorial plate, MxB forms two patches that are co-localized with the centrosome markers Aurora A and γ-tubulin. We are investigating which NPC proteins (nucleoporins) are also localized to the centrosome, how MxB affects the function of the centrosome as a microtubule-organizing center, and what is the potential impact of MxB on the trafficking of viral DNA along microtubules.

Association of NELF with RNAPII induces a transcriptional pause shortly after promoter clearance, an important step in the regulation of gene expression. This promoter-proximal pause is also important for the pathogenesis of the human immunodeficiency virus (HIV). In the context of an active infection, the viral transactivator of transcription (Tat) protein stimulates viral transcription elongation by recruiting P-TEFb, effectively overcoming the promoter-proximal pause. One mechanism for HIV latency can involve low levels of Tat in the infected cell that results in inefficient recruitment of P-TEFb to the paused RNAPII. This leads to a prolonged promoter-proximal pause on the provirus and entry of the provirus into transcriptionally latent state. These latently infected cells create viral reservoirs, which are not targeted by T cell responses or antiretroviral drugs due to minimal viral RNA transcription and protein translation. Therefore, latently infected cells remain hidden until transcription of the provirus is induced.
The hepatitis delta small antigen (HDAg-S), one of the two proteins produced by the hepatitis delta virus (HDV), stimulates RNAPII processivity during HDV transcription and is thought to do so by competing with NELF for a common binding site on RNAPII. The aim of this study is to evaluate the anti-latency potential of inhibiting the RNAPII pausing using a NELF competitor such as HDAg-S. As the promoter-proximal pause is one of the mechanisms through which HIV becomes latent, using a NELF competitor could lead to transcriptional reactivation of latently infected cells. We report that HDAg-S stimulates transcription of latent HIV provirus mutants in infected cell lines. Interestingly, our results hint at a synergistic relationship between Tat and HDAg-S, one in which HDAg-S kickstarts viral transcription, inducing a positive feedback loop that stimulates Tat production. These results demonstrate the anti-latency potential of inhibiting the promoter-proximal pause using a NELF competitor.

The persistent latent reservoir of long-lived cells carrying integrated HIV DNA is the source of reinfection upon treatment interruption, and a primary focus for cure research. The reservoir is difficult to study because these cells are relatively rare or located in tissues that are difficult to sample. Sequencing proviral DNA in the latent reservoir is an important source of information about reservoir establishment and persistence, especially from the presence of identical (clonal) sequences. We evaluated the relationship between clonality measures and drivers of reservoir persistence, e.g., clonal expansion, by implementing a simulation model of within-host HIV dynamics in actively and latently infected cells. We implemented a discrete event simulation in the R package treeswithintrees, with four populations of cells corresponding to active, latent, replenishment and death compartments. We used Latin Cube Hypersampling to generate parameter setting combinations to ensure sufficient coverage across all realistic parameter values. To simulate molecular evolution on the resulting trees, we collapsed branches representing infected cells in a latent state and ran the program INDELible with parameters calibrated to HIV-1 on a representative env sequence. We evaluated three clonality measures: the proportion of identical sequences, Gini coefficient, and number of identical pairwise comparisons. We determined that the Gini coefficient was the least responsive to variation in rates of clonal expansion. In addition, we found that the interpretation of clonality measures is contingent on other model parameters that influence the baseline probability of sampling identical sequences.
Our results caution against relying on the Gini coefficient and proportional of identical sequences when characterizing the latent reservoir. Future work includes testing this approach using real world HIV data.

HIV-1 assembly is dependent on the expression of structural proteins (Gag/Pol) and their dynamic interactions with the RNA genome. Similar to other retroviruses, the HIV-1 genome is integrated into host cell chromosomal DNA within the nucleus. It is well-established that HIV-1 encodes the Rev protein to facilitate the nuclear export of intron-containing Gag/Pol mRNA. Once within the cytoplasm, additional evidence suggests Rev further enhances Gag/Pol mRNA translation and Gag protein-RNA interaction as part of virion assembly. Yet, the mechanism underpinning Rev-RNA complex cytoplasmic localization remains poorly understood. Additionally, the molecular driver responsible for directing the Rev-RNA complex to different sites of translation and assembly has not been elucidated. Recently, a membrane sorting protein in the endolysosomal pathway, PACS-1, was demonstrated to interact with HIV-1 Rev. Thus, we hypothesize HIV-1 Rev hijacks PACS-1 to serve as a molecular driver to enhance protein translation and virion assembly. Herein, we utilized bimolecular fluorescence complementation and confocal microscopy, to demonstrate that Rev interacts with PACS-1 within the perinuclear region. Furthermore, the Rev and PACS-1 interaction decreased in the presence of Rev mutants deficient in RNA binding, oligomerization, or nuclear export. Upon HIV-1 infection of CD4+ HeLa cells, we observed that PACS-1 co-localized with HIV-1 Rev across various subcellular locations, including ribosomes and endosomes. In particular, the strength of co-localization was dependent on distinct stages of the viral replication cycle. Overall, this research presents an important regulatory pathway during HIV-1 assembly for future therapeutic targets and provides novel insights into the mechanism of RNA subcellular trafficking.

Human immunodeficiency virus-1 (HIV-1) is the main cause of acquired immunodeficiency syndrome (AIDS) and is transmitted by contact with infected fluids, including genital secretions (during sex) or blood. As a result, HIV-risk groups include heterosexual individuals (HET), men-who-have-sex-with-men (MSM), people who inject drugs (PWID) and people who received contaminated blood transfusions (CBT). When HIV-1 is transmitted, typically only a single clone or in rare cases a few HIV-1 clones establish the new infection, representing a transmission bottleneck for the virus. The viral clone that establishes infection is called transmitted/founder (T/F) virus. The phenomenon is especially outstanding in HET and MSM transmission while less stringent in transmission from blood contact (PWID and CBT). Specific traits that allow for successful transmission have not been identified clearly. This project determined the transmission fitness between T/F viruses from different transmission routes by using in vitro competition and then analyzed the contribution of possible factors by using a series of phenotypic assays. We found that T/F viruses from HET and MSM often outcompete T/F viruses from PWID during in vitro competitions on T helper type 1 cell line while PWID will dominate the infection on T helper type 17 cell line. When it comes to phenotypic factors, T/F viruses require more stringent cellular co-receptor conformation to enter susceptible cells compared with chronic viruses, especially viruses from HET. Furthermore, T/F viruses across all transmission modes show faster cell entry speed than that of chronic viruses. This project will establish key viral phenotypes contributing the successful virus transmission and entry and will contribute to the design of a robust and protective anti-HIV vaccine.